Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application

The lack of an efficient, low-cost sequencing method has long been a significant bottleneck in protein research and applications. In recent years, the nanopore platform has emerged as a fast and inexpensive method for single-molecule nucleic acid sequencing, but attempts to apply it to protein/peptide sequencing have resulted in limited success. Here we report a strategy to control peptide translocation through the MspA nanopore, which could serve as the first step toward strand peptide sequencing. By conjugating the target peptide to a helicase-regulated handle-ssDNA, we achieved a read length of up to 17 amino acids (aa) and demonstrated the feasibility of distinguishing between amino acid residues of different charges or between different phosphorylation sites. Further improvement of resolution may require engineering MspA-M2 to reduce its constriction zone's size and stretch the target peptide inside the nanopore to minimize random thermal motion. We believe that our method in this study can significantly accelerate the development and commercialization of nanopore-based peptide sequencing technologies.

by Qitan Technology. 3 In the case of helicase dimer, we connect two MTA helicase monomers head -to-tail with a linker of (AHIVMVDAYKPT). The gene was constructed to pET-15b vector with 7 consecutive histidine on the N-terminal of protein sequence to facilitate the purification.
The transformation and expression of the helicase protein, both monomer and dimer, were the same as the above MspA protein except the use of antibiotics. After expression, the bacteria cell pellets were resuspended in Buffer (50 mM Tris-HCl, 500 mM NaCl, 1 mM DTT, 1 mM EDTA, 5%glycerol, pH 8.0), and lysed with high pressure homogenizer. After centrifuge, the supernatant was added with 60% ammonium sulfate and incubated at 4 °C for 2 hours. The salting-out solution was centrifuged at 18000 g for 30 min, and the precipitated pallets were dissolved in 20-30 mL of buffer (50 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 5%glycerol,pH 8.0). The solution was loaded on the Ni-NTA column, washed with 15 mM imidazole and eluted with 250 mM imidazole. Then, the sample was dialysis and further purified with Heparin column and SD 200 size exclusion column. The purified sample was verified by SDS-PAGE (Fig. S1b). Briefly, 100 nM of pre-annealed b+d dsDNA and 1 μM oligo f (Table S1) were mixed in solution (5 mM HEPES, 5 mM ATP, 5 mM MgCl 2 , and KCl with different concentration). Then, MTA helicase monomer or dimer was added to reach final concentration of 100 nM. The fluorescent signal is monitored under 488 nm excitation and 515 nm emission setup (Fig. S2b).

Verify the
The binding affinity of helicase to ssDNA is studied with fluorescent polarization methods. 4 According to literatures, we synthesized the 44 nt oligo g with a Hexachloro fluorescein (HEX) attached to the thymine base at position 37. The MTA helicase monomer or dimer was bufferexchanged to 50 mM HEPES pH 8.0 solution with different salt concentration. To start the measurement, 0.78 nM to 800 nM helicase was mixed with 1 nM oligo g, and the fluorescent anisotropy was assessed over the course of 20 min at 25 °C.

4.
Conjugation of oligo peptide with ssDNA. As a model system, the oligo peptide Table S3 used in this study was designed and synthesized with N-terminal azide and C-termial cysteine. The handle-ssDNA including 8 different sequences are shown in Table S2. In this way, different peptide can be tested together in one nanopore experiment and separated from each other by the handle-ssDNA signal, which can largely improve the sequencing throughput. The handle-ssDNA were synthesized with DBCO group at 5'end, and the lead-ssDNA with poly-T sequence was synthesized with maleimide group at the 3'end (Fig.S3a). The conjugation reaction was conducted in 1x PBS buffer with handle: peptide: lead ratio of 1:2:1. The reaction was shaken at 30 °C for 18 hours. The conjugation product was verified by 8% 7 M urea polyacrylamide denatured gel electrophoresis

Electrophysiology recordings and data analysis.
All electrophysiology measurements were performed according to literature. 5 Briefly, a measurement chamber is consisted of two compartments separated by a plastic tube with an orifice (~25 µm in diameter). Before the measurement, the compartments were first cleaned with isopropanol, H 2 O 2 and Milli-Q water, followed by drying with compressed nitrogen gas. A pair of Ag/AgCl electrodes purchased from Warner were inserted in each compartment to conduct current. In principle, the compartment that is electrically grounded is defined as the cis and the opposing side is defined as the trans side. The orifice on the tube was then treated with 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti Polar Lipids) in hexane, and leave for drying. Approximately 0.1 mL of electrolyte buffer (10 mM HEPES, 400 mM KCl, 5 mM Mg 2+ , pH 8.0) was added to both compartments. We use a pipette to form and drag an air bubble across the orifice, which will introduce a self-assembled phospholipid bilayer across the orifice. MspA-M2 nanopores protein were diluted and added to cis chamber to obtain spontaneous pore insertions. The cis chamber was immediately perfused with fresh buffer upon the signal of single-pore insertion was obtained.
To prepare the sequencing solution, the peptide DNA conjugate was firstly annealed with a cholesterol-tagged ssDNA oligo (tetheroligo), and then combined with 500 nM MTA helicase solution, before added to the cis chamber. Under 180 mV applied voltage, the sequencing is initiated by addition of 2 µL 100 mM ATP (Thermo Fisher) to the cis chamber. The electrophysiology signals were amplified by an Axopatch 200B patch clamp amplifier and digitized by a Digidata 1550B digital-to-analog converter (Molecular Devices). The ionic current signal is sampled with a 25 kHz sampling rate and the output was filtered at a 1 kHz angular frequency. Some of the experiments were performed with a homemade trans-impedance amplifier paired with a digitizer from National Instrument. Except the study of the upward-spike phenomenon, all the raw data was filtered with an additional 8-pole Bessel-filter at 100 Hz before analysis and plotting.
The sequencing experiment was typically performed for 10 minutes to 1 hour, during which the solution evaporation or the temperature changes could affect the nanopore system causes some              As a result, no significant changes of translocation time is observed. It is possible that the electrical field out of the trans exit quickly attenuated, thus only the nearest nucleotides can contribute to tugging force, so that the length of the poly-T may not affect the overall pulling back process.